Transformation of plant cells

ABSTRACT

Plant cells are transformed by bringing them into contact with a a multiplicity of needle-like bodies on which the cells may be impaled. This causes a rupture in the cell wall allowing entry of transforming DNA either from a surrounding liquid medium or of DNA previously bound to or otherwise entrapped in the needle-like projections.

This is a continuation-in-part of application Ser. No. 07/541,890, filedJun. 21, 1990, now abandoned the entire contents of which isincorporated herein by reference.

This invention relates to a method of transforming plant cells.

The choice of method for the transformation of plant cells tends to belimited to those which are convenient for the target plant type. As ageneralisation, dicotyledonous plants are relatively easy to transformwhereas monocotyledonous plants are very difficult, there being only afew techniques available in respect of which success has been reported,and that with very low success rate. There is, therefore, a need to makeavailable new techniques for transformation of monocotyledonous plants,which group includes the agronomically most important cereal crops.

One method which is claimed to transform cereal plant cells is theprocedure known as "microinjection" where, under the microscope, a DNAconstruct is injected from a hollow needle into a target cell. A variantof that procedure is the rupturing of the cell wall with a needle, theDNA being added to the surrounding medium and allowed to diffuse intothe cell through the break in the cell wall. This is known as"micropricking". Both of these procedures require a high degree ofmanipulative skill by the operator and are very time consuming.

An alternative approach which has been proposed abandons the highprecision of targeting which is inherent in microinjection andmicropricking, in favour of a rapid "pepperpot" approach which enableslarge numbers of cells to be "hit" in a short time, giving a largenumber of putative transformants for screening.

In one such approach, solid particles, such as tungsten or goldmicrospheres, bearing surface-bonded DNA are fired at target tissue atvery high velocity, for example under propulsion of an explosive charge.One problem with this technique is the effect of the blast of expandinggas on the target tissue. Another is the difficulty of aiming theprojectile shower at a selected area of the target.

An object of the present invention is to provide an improved method fortransformation of target cells, particularly plant cells.

According to the present invention there is provided a method oftransforming cells comprising contacting the cells with a multiplicityof needle-like bodies so that the cells are impaled upon the saidbodies, transforming DNA being either surface-bound to the projectionsor present in a liquid medium in contact therewith.

In one embodiment of the invention a quantity of the needle-like bodiesis added to a liquid suspension of the cells to be transformed and themixture agitated, for example by stirring, so that the moving cells andbodies interact resulting in penetration of the cell wall of the cells.

One method comprises mixing the DNA and fibre suspension, then addingthis mixture to the cell suspension. The final mixture is vortexedtogether. The cells can then be incubated, and tested for expression ofrecombinant DNA. It has been found that the efficiency of DNA deliveryvaries according to the conditions; it is affected by several factorsincluding the following: vortex time; cell suspension type (variationalso found by H. F. Kaeppler et al, 1990, Plant Cell Reports, 9,415-418); cell suspension age; osmolarity of culture medium; type offibres; number of fibres present; type of DNA construct; concentrationof DNA. Other factors which may affect DNA delivery include: thephysical mixing methods used; the size, shape and uniformity of thefibres; the topology of the DNA (e.g. linear, supercoiled); the presenceof "carrier" DNA alongside the transforming DNA.

In another embodiment of the invention, the impalement of the cells onthe projections may be effected under a slowly increasing, andcontrolled, mechanical force which urges the cells on to a surfacebearing upstanding needles-like projections. Force may be applied bycompressed gas or by a piston arrangement or by centrifuging the cellson to the said surface.

The surface bearing the needle-like projections may be porous and thecells may be drawn into contact therewith by the application of anattractive force on the remote side of the surface, such as a shockevacuation.

A further method comprises attracting the cells to be transformed on tosurface bonded needle-like projections by an attractive electric field.Once located proximate the projections, impalement may be effected by,for example, application of a high-frequency alternating field or,alternatively, by causing an electric discharge from the needle-likeprojections, which are ideal for creating discharges, to perforate thecells.

Once the cells have been impaled on the projections, they may beretained there for whatever is found to be the optimum time interval toeffect entry of the DNA into the cells: thereafter they may be repelledfrom the projections into a culture medium by application of an electricfield of suitable polarity and strength, and the wound allowed to closeand cells allowed to replicate.

Yet another method of effecting impalement is to provide a magneticcarrier material to which the cells may be attached and to draw thecells onto the needle-like projections using an attractive magneticforce. The magnetic cells, once drawn onto or near to themicroprojections may be treated as described for electric fieldattraction.

Possible needle-like materials for use in this invention are (a) hollowfibres, in the bore of which DNA may be trapped, which may besurface-bonded in upstanding configuration in the form of a felt or"velvet" type of fabric (b) a metallic plate bearing metallic dendriticcrystal growths (c) setacious metals (d) a metal or ceramic whiskerswhich may be in the form of a mat thereof in which the whiskers may berandomly oriented but with a sufficient number presenting a sharp end tothe cells (e) carbon, silicon carbide and like fibres (f) glass fibresand, (g) other elongate crystalline materials. Suitable surface bondedforms may be produced by known techniques such as tufting or flockingwhich utilize an electrostatic charge to cause fibres to stand on end ona substrate to which they may be attached.

Many fine fibrous materials are known which may be used in thisapplication, for example alumina, silica, titania, zirconia, boron,carbon, compounds such as the carbides, and glass fibre. For use in thisinvention they may be coated with a biocompatible coating to preventdisadvantageous effects. Indeed the provision of such a coating may beadvantageous in that it may provide a surface with which transformingDNA may be more conveniently bound, thus improving delivery of the DNAto the interior of the cell. Suitable such coatings may be syntheticresinous materials, surfactants and soluble benign materials such asalginate or gelatin.

In fundamental principle, this type of transformation utilises aprocedure which penetrates the cell wall in a non-lethal manner. Suchmethods, then, seek to wound but not kill the cells. In investigatingpossible procedures, it may be assumed that if particular method iscapable of killing the cells then by making the treatment less severethe method may be adapted to wound.

The invention will now be described, by way of illustration, in thefollowing Examples.

EXAMPLE 1 Viability of Cells After Fibre Treatment

Two grades of silicon carbide whiskers were used in this Example, 30×0.5μm and 10×0.3 μm. The whisker samples were washed in three changes ofwater, sterilised in ethanol and air-dried. Finally the whiskers wereplaced in a cell culture medium, approximately 100 mg of whiskers per 10ml of medium, and used as a suspension.

Two suspension maize cell lines, one designated LO56 and the other BlackMexican Sweet corn (BMS), were assessed with respect to the effect ofthe whiskers on cell viability following vortexing in the presence ofthe whiskers. A degree of cell mortality in this test is indicative ofsuccessful treatment of those cells which survive (similarconsiderations apply in other techniques such as electroporation).

Initial experiments were performed using LO56 cells in a total volume of1.75 ml of cell suspension, whiskers and medium (1.0 ml cell suspension,3 days post culture; 0.5 ml sterile distilled water, to reduceosmolality and 0.25 ml of whisker suspension) using two vortexdurations, 90 and 180 seconds. Vortexing was conducted using a standardGallenkamp (Trade Mark) laboratory vortex mixer in either 10 or 50 mlsterile plastic centrifuge tubes.

Observation of the LO56 cells immediately after treatment suggested thatno significant effect on cell integrity had occurred using any of thetreatments. FDA viability testing after 20 hours confirmed that this wasthe case. From previous experience of LO56 in other contexts we wereaware that it has particularly rigid cells walls.

The same parameters, i.e. vortex duration and tube size were then testedwith BMS cells (one day after sub-culture). The viability of thesecells, which are considerably larger than LO56, showed a clearrelationship with vortex duration using the 50 ml tubes for mixing. Withthe 180 second treatment approximately half of the cells were found tohave been damaged after 20 hours as determined by FDA fluorescence,compared with the controls. Again, relatively little effect was seenusing the 10 ml tubes. This confirms the need for the greater degree ofmixing which is permitted by the larger diameter tubes.

A clear association was seen between the cells and whiskers immediatelyafter treatment, with the silicon carbide whiskers being particularlyconcentrated in the intercellular spaces of cell clusters. Whilstwhiskers were seen on the surface of some cells their narrow diametermade it impossible to judge whether cell wall penetration had occurred.

EXAMPLE 2 Demonstration of Transient Expression of DNA AfterFibre-Mediated Transformation

In order to demonstrate that DNA can be inserted by this method, afurther experiment was carried out using BMS and controls and treatmentslisted in the Table below. Cell viability was measured after 20 hoursand a fluorimetric GUS assay after 40 hours after treatment. The valuesobtained are also shown in Table 1. The most significant result is thatfor treatment T4 in which GUS expression was twice that of thebackground (control C5).

                  TABLE 1                                                         ______________________________________                                        Test  Whiskers DNA      Vortex                                                                              Time  Viability                                                                            GUS                                ______________________________________                                        C1    -        -        +           82     2.3                                C2    -        +        +     90    75     1.1                                C3    +        -        -           72     2.0                                C4    +        -        +     90    74     2.0                                C5    +        -        +     180   43     2.0                                C6    +        -        -           65     2.4                                T1    +        +        +     90    68     1.0                                T2    +        +        +     180   59     1.6                                T3    +        +        +     90    75     1.5                                T4    +        +        +     180   33     4.2                                ______________________________________                                    

Tests C1 to C6 are controls and T1 to T4 are the tests of the method ofthis invention.

A mixture of both the 30×0.5 μm and the 10×0.3 μm silicon carbidewhiskers was used in these tests.

DNA was used in the form of a CaMV35S/GUS construct. Cell Viability wasmeasured after 20 hours with FDA and GUS expression is in units offluorescence/hour/μg protein.

EXAMPLE 3 Fibre-Mediated Transformation

Further experiments to demonstrate fibre-mediated transformation wereperformed.

A suspension maize cell line designated Black Mexican Sweetcorn (BMS)was used. The suspension was subcultured once a week onto fresh BMSmedium containing Murashige and Skoog medium, 2% sucrose, 2 mg/ml 2,4-Dand adjusted to pH 5.6 prior to filter sterilising.

Silicon carbide fibres (30 μm×0.5 μm) were sterilised under ultravioletlight, then suspended in sterile water to give a 5% solution.

The DNA construct used was a plasmid designated pAI₁ gusN, containingthe Adh 1 promoter and the β-glucuronidase (GUS) reporter gene. The DNAwas in the supercoiled form.

A standard GUS fluorometric assay was used to evaluate the amount of DNAtaken up by the cells, and was carried out 48 hours after fibretreatment of the cells. The fluorometric assay used the substrate4-methyl umbelliferyl glucuronide (NUG) which is cleaved by GUS torelease a fluorescent product (4-Mu), measured by a fluorometer. ABradford protein assay was used to calculate GUS expression, based onstandards and controls.

Transformation was carried out as follows: 75 μg of plasmid DNA wasvortexed for 10 seconds with 80 μl of the fibre suspension. A cellsuspension, consisting of 250 μl of packed 3 day-old BMS cellsresuspended in 100 μl sterile water with an osmolarity of 125 mOsM/kg,was then added to the DNA/fibre suspension. This mixture was vortexedtogether for 60 seconds using a standard desktop vortex mixer at thehighest possible speed. The conditions used were those found to beoptimal for transforming BMS cells.

After vortexing, the mixture was distributed into welled trays to which1 ml of conditioned media was added. The trays were incubated at 25° C.for 48 hours before GUS assays were performed.

Four replications for each suspension were tested, and four controlswere used:

C1: the untreated cell suspension culture;

c2: cell suspension/fibres/DNA without vortexing;

C3: cell suspension/DNA vortexed without fibres;

C4: vortexed cell suspension/fibres/DNA mixture containing anegative-GUS construct (pCaI₁ catN, containing the CaMV35S promoter andthe CAT reporter gene).

All experiments were carried out under sterile conditions.

Results are given in Table 2, and show that the treated cells areexpressing the GUS gene.

                  TABLE 2                                                         ______________________________________                                        MEAN GUS    MEAN GUS        INCREASE OF                                       EXPRESSION  EXPRESSION      TREATED                                           OF CONTROLS OF TREATED CELLS                                                                              CELLS OVER                                        (μmol 4 Mu/mg/hr)                                                                      (μmol 4 Mu/mg/hr)                                                                          BACKGROUND                                        ______________________________________                                        0.0518      1.5417          29.7X                                             ______________________________________                                    

EXAMPLE 4 Effect of Varying Conditions on Fibre-Mediated Transformation

Several experiments were carried out to investigate the conditions whichenable successful DNA delivery to cells. The method and conditionsdescribed in Example 3 were used, unless otherwise stated. Differentfactors were varied as follows:

a) Vortex time was varied when mixing the cell/DNA/fibre suspension. Forthese experiments, the plasmid was pCaI₁ gusN (containing the CaMV35Spromoter and the GUS gene), and the method used 25 μg DNA, 40 μl fibresuspension, and 5 day-old cells resuspended in BMS medium (osmolarity of202 mOsm/kg). Results are shown in Table 3. The controls gave a meanbackground GUS expression of 0.00693 μmol 4 Mu/μg protein/hour.

                  TABLE 3                                                         ______________________________________                                                                   INCREASE OF                                        VORTEX  MEAN GUS EXPRESSION                                                                              TREATED                                            TIME    OF TREATED CELLS   CELLS OVER                                         (seconds)                                                                             (μmol 4 Mu/mg/hr)                                                                             BACKGROUND                                         ______________________________________                                         60     0.0721             10.5X                                              120     0.0504             7.3X                                               240     0.0326             5.0X                                               ______________________________________                                    

b) Cell cultures of different ages (at different growth stages) wereused. For these experiments, the plasmid was pCaI₁ gusN, and the methodused 25 μg DNA, 40 μl fibre suspension, and cells resuspended in BMSmedium. Results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                               MEAN GUS     MEAN GUS                                                         EXPRESSION   EXPRESSION  INCREASE                                      AGE OF OF CONTROLS  OF TREATED  OF TREATED                                    CELLS  (μmol 4 Mu/                                                                             CELLS (μmol                                                                            CELLS OVER                                    (days) mg/hr)       4 Mu/mg/hr) BACKGROUND                                    ______________________________________                                        3      0.0333       1.01        30X                                           5      0.0064       0.12        19X                                           7      0.0286       0.7055      24X                                           10     0.0110       0.2358      21X                                           ______________________________________                                    

c) The volume of the 5% fibre suspension used in the mixture was varied.For these experiments, the method used 25 μg DNA and cells resuspendedin BMS medium. Results are shown in Table 5. The controls gave a meanbackground GUS expression of 0.0261 μmol 4Mu/μg protein/hour.

                  TABLE 5                                                         ______________________________________                                        VOLUME OF MEAN GUS        INCREASE OF                                         FIBRE     EXPRESSION      TREATED                                             SUSPENSION                                                                              OF TREATED CELLS                                                                              CELLS OVER                                          (μl)   (μmol 4 Mu/mg/hr)                                                                          BACKGROUND                                          ______________________________________                                         80       1.0314          39.5X                                                90       0.8242          31.6X                                               100       0.7928          30.4X                                               120       0.0783           3.0X                                               ______________________________________                                    

d) The DNA concentration used in the mixture was varied. For theseexperiments, the method used cells resuspended in BMS medium. Resultsare shown in Table 6. The controls gave a mean background GUS expressionof 0.0580 μmol 4Mu/μg protein/hour.

                  TABLE 6                                                         ______________________________________                                                  MEAN GUS        INCREASE OF                                                   EXPRESSION      TREATED                                             AMOUNT    OF TREATED CELLS                                                                              CELLS OVER                                          OF DNA (μg)                                                                          (μmol 4 Mu/mg/hr)                                                                          BACKGROUND                                          ______________________________________                                        25        1.001           17.3X                                               50        1.467           25.4X                                               75        1.625           28.1X                                               ______________________________________                                    

e) The osmolarity of the culture medium (affecting cell turgidity) usedin the mixture was varied. The cell culture was resuspended in BMSmedium (202 mOsM/kg), a 1:3 mixture of BMS and water (164 mOsM/kg), orin sterile water (125 mOsM/kg). Results are shown in Table 7. Thecontrols gave a mean background GUS expression of 0.0175 μmol 4Mu/μgprotein/hour.

                  TABLE 7                                                         ______________________________________                                        OSMOLARITY MEAN GUS        INCREASE OF                                        OF CULTURE EXPRESSION      TREATED                                            MEDIUM     OF TREATED CELLS                                                                              CELLS OVER                                         (mOsM/kg)  (μmol 4 Mu/mg/hr)                                                                          BACKGROUND                                         ______________________________________                                        202        0.0932           5X                                                164        0.1831          10X                                                125        0.2142          12X                                                ______________________________________                                    

The results in Tables 3-7 show that transient expression is successfullyachieved under a variety of conditions.

We claim:
 1. A method of introducing a nucleic acid into plant cellscomprising providing in a liquid medium (i) plant cells suspendedtherein, (ii) a multiplicity of metal or ceramic whiskers bodies and(iii) a nucleic acid, and subjecting said liquid medium containing thesaid suspended cells, the said metal or ceramic whiskers bodies and saidnucleic acid to physical motion so as to create collisions between saidmetal or ceramic whiskers and said plant cells whereby said nucleic acidis introduced into said plant cells.
 2. A method as claimed in claim 1in which the cells are cells of a monocotyledonous plant.
 3. The methodaccording to claim 1 wherein said plant cells are capable ofregeneration into whole plants.
 4. The method according to claim 3wherein said plant cells are regenerable cells of Zea mays.
 5. A methodfor introducing a nucleic acid into regenerable cells of Zea mayscomprising providing in a liquid medium (i) said regenerable cells ofZea mays suspended therein, (ii) a multiplicity of silicon carbidewhiskers, and (iii) a nucleic acid, and subjecting the said liquidmedium containing the said suspended regenerable cells of Zea mays, thesaid silicon carbide whiskers and said nucleic acid to physical motionso as to create collisions between said silicon carbide whiskers andsaid suspended regenerable cells of Zea mays whereby said nucleic acidis introduced into said regenerable cells of Zea mays.
 6. The method ofclaim 5 wherein said nucleic acid is a DNA.